The present invention, in some embodiments thereof, relates to molecular biology and, more particularly, but not exclusively, to detection and mapping of 5-hydroxymethyl cytosine bases within single or plurality of DNA molecules.
Epigenetics refers to DNA and chromatic modifications that persist from one cell division to the next without change in the underlying DNA sequence. These dynamic, chemical modifications are a major source of genomic variation, yet this variation is difficult to detect by current technologies since it is masked by ensemble averaging.
Epigenetic modifications include cytosine methylation (5mC) and the recently discovered cytosine hydroxymethylation (5hmC), which exhibits tissue and cell type specific distribution in mammalian genomes.
In recent studies of genomic DNA from human and mouse brain tissue and mouse embryonic stem cells, it was found that a substantial fraction of 5-methylcytosine (5mC) in CpG dinucleotides is converted to 5hmC by the action of the Tet family Fe(II)-dependent oxygenases. The distribution of 5hmC in mammals is tissue specific and non-random, suggesting that its deposition is highly regulated and that it may have a functional role in transcription regulation. Today, 5hmC is widely accepted as the sixth base of DNA (after 5-methylcytosine, the fifth base), and it is in the focus of extensive research.
To elucidate the role of 5hmC, information regarding quantity and distribution is critical, and several methods for the specific detection of 5hmC have been reported since its discovery in mammalian tissue in 2009 [M. Münzel, D. Globisch, and T. Carell, Angewandte Chemie (International ed. in English), 2011, 50, 6460-8].
Selective functionalization of 5hmC is based on the discovery that T4 β-glucosyltransferase (β-GT) from T-4 bacteriophages can attach a glucose moiety from uridine diphosphoglucose (UDP-Glu) onto the hydroxyl group of 5hmC, resulting in a glucosylated nucleotide. Song et al. [in Nature biotechnology, 2011, 29, 68-72 and U.S. Patent Application having Publication No. 2011/0301045] utilized this enzymatic process to transfer a glucose chemically modified with an azide group onto 5hmC in genomic DNA. Using Huisgen cycloaddition (click) chemistry, they attached a biotin to the azide group and captured the 5hmC-containing DNA on streptavidin-coated magnetic beads for sequencing. A commercially available product, the Hydroxymethyl Collector™ (by Active Motif), for detecting and capturing DNA fragments containing 5-hmC methylation, was developed based on this methodology.
However, due to the short sequence reads, sequencing reports on the population averaged distribution of 5hmC cannot resolve small sub-populations or characterize variation in the 5hmC patterns, which are required for identifying epigenetic modifications which may display high cell to cell variation due to their dynamic nature.